Nucleotide integrases are molecular complexes that are capable of cleaving nucleic acid substrates at specific recognition sites and of concomitantly inserting nucleic acid molecules into the nucleic acid substrate at the cleavage site. Thus, nucleotide integrases are useful tools, particularly for genome mapping, genetic engineering and disrupting the synthesis of gene products. Structurally, nucleotide integrases are ribonucleoprotein (RNP) particles that comprise an excised, group II intron RNA and a group II intron-encoded protein, which is bound to the group II intron RNA.
Conventionally, nucleotide integrases are made by isolating RNP particles that have nucleotide integrase activity from source organisms which comprise a DNA molecule that encodes both the RNA and protein subunits of the nucleotide integrase. In order to obtain nucleotide integrases other than wild type, the source organisms are mutagenized. The mutagenesis is a laborious, multistep process. Moreover, this process yields limited quantities of the nucleotide integrase.
Accordingly, it is desirable to have methods for making nucleotide integrases which are not laborious and which permit the nucleotide integrase to be readily modified from the wild type. Methods which yield at least microgram quantities of substantially pure nucleotide integrases are especially desirable.